Development and evaluation of a new immunohistochemistry-based test for the detection of rabies virus neutralizing antibodies

Abstract

Rabies claims about 55,000 human lives and many hundreds of thousands of livestock every year, worldwide. Despite a heavy disease burden, laboratory facilities to diagnose the infection remain scarce in most countries of the developing world where the disease is endemic. Rapid Fluorescent Focus Inhibition Test (RFFIT) and Fluorescent Antibody Virus Neutralization Test (FAVN) are the common tests done in the rabies diagnostic laboratories to detect and quantitate Rabies Virus Neutralizing Antibodies (RVNA). RFFIT is most often employed in confirming seroconversion following prophylactic vaccination, and to aid ante-mortem diagnosis in suspected cases of rabies. Though this remains one of the most sought-after diagnostic services in rabies laboratories, the requirements for expensive anti-rabies fluorochrome antibody conjugate and a fluorescent microscope restrict its performance to only a few reference laboratories. Cost-effective laboratory diagnostic methods employing affordable technology are a need of the hour in the rabies-endemic countries. In this study we have developed a new immunohistochemistry-based neutralization test and extensively evaluated it along with RFFIT. One hundred and 20 human serum samples collected after post-exposure vaccination were subjected to both the tests for determining RVNA titers. The results obtained with the new test correlated significantly with those of RFFIT. Further validation of the inter- and intra- assay precision, lower limit of quantification (LLOQ) and specificity was also performed. The best correlation between the 2 methods, however, was observed only when the RVNA concentrations in the samples were>20 IU/mL. Overall, the immunohistrochemistry-based neutralization test yielded satisfactory results. We suggest that it might serve as a cost-effective alternative to RFFIT in low-resource settings in the developing countries.

Keywords: RFFIT; immunohistochemistry; post-exposure prophylaxis; rabies; rabies virus neutralizing antibodies.

Figure 1. Showing the 50% end point dilution in the Rapid Fluorescent Focus Inhibition Test. Note approximately 50% BHK 21 cells in the microscopic field are infected as evidenced by presence of fluorescent foci representing rabies nucleoprotein (×400).

Figure 2. Showing 50% end point dilution in the immunohistochemistry based test. Note the presence of dark brown inclusions representing rabies nucleoprotein in approximately 50% of BHK 21 cells (×400).

Figure 3. Bland Atman plot showing the agreement between the 2 tests. Please see section “results” for explanation.

Figure 4. Scatter plot graph of results obtained between the 2 tests. Note the good correlation among samples with high titers (>20 IU/mL). There was no correlation with samples having titer less than 20 IU/mL.